The mechanism by which DNA helicases unwind double-stranded DNA will be examined. DNA helicases mediate DNA replication, repair, recombination, and transcription through the coupling of nucleotide hydrolysis to translocation on single-stranded DNA and separation of the strands of double-stranded DNA. In the research proposed here, the gene 4 protein encoded by T7 bacteriophage will be used as a model helicase. T7 gene 4 protein is a replicative DNA helicase that binds single-stranded DNA as a hexamer and, as it translocates in the 5' - to 3'- direction, unwinds double-stranded DNA using the energy obtained from the hydrolysis of dTTP. This research will focus on site-directed mutagenesis of specific conserved amino acids and biochemical analysis of the resulting genetically altered proteins. Although DNA helicases including T7 gene 4 protein have been characterized biochemically, there is little known about the catalytic mechanism and the amino acids involved.